Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern Queensland, Australia.

Ninety-three giant Queensland grouper, Epinephelus lanceolatus (Bloch), were found dead in Queensland, Australia, from 2007 to 2011. Most dead fish occurred in northern Queensland, with a peak of mortalities in Cairns in June 2008. In 2009, sick wild fish including giant sea catfish, Arius thalassinus (Rüppell), and javelin grunter, Pomadasys kaakan (Cuvier), also occurred in Cairns. In 2009 and 2010, two disease epizootics involving wild stingrays occurred at Sea World marine aquarium. Necropsy, histopathology, bacteriology and PCR determined that the cause of deaths of 12 giant Queensland grouper, three wild fish, six estuary rays, Dasyatis fluviorum (Ogilby), one mangrove whipray, Himantura granulata (Macleay), and one eastern shovelnose ray, Aptychotrema rostrata (Shaw), was Streptococcus agalactiae septicaemia. Biochemical testing of 34 S. agalactiae isolates from giant Queensland grouper, wild fish and stingrays showed all had identical biochemical profiles. The 16S rRNA gene sequences of isolates confirmed all isolates were S. agalactiae; genotyping of selected S. agalactiae isolates showed the isolates from giant Queensland grouper were serotype Ib, whereas isolates from wild fish and stingrays closely resembled serotype II. This is the first report of S. agalactiae from wild giant Queensland grouper and other wild tropical fish and stingray species in Queensland, Australia.

Artelon as a Bio-Scaffold to Augment Collateral Ligament Repair after Knee Dislocation.

Introduction: Knee dislocations (KD) have high rates of multi-ligamentous injury (MLI). Collateral ligaments rupture in 50-60% of KDs. Traditionally, collateral ligaments have undergone primary repair, though microscopic healing is not optimal. Artelon is a degradable, polyurethane urea bio-scaffold thought to decrease mechanical forces and promote healing, motion, and strength. Currently, little evidence exists regarding its indications or outcomes. Material and methods: Thirty-two patients with KD and MLI undergoing collateral ligament repair at a level-I trauma centre between 2015-2020 were included. Patients age <18, with ipsilateral fractures or inadequate follow-up were excluded. The Artelon (AG) and primary ligamentous repair group (PR) each included 16 patients. Injury and perioperative variables were evaluated using SPSS® . Results: Thirty-two KDs were included in 32 patients, with 60% anterior. There were no significant differences between the two cohorts demographically or with regards to the type or severity of injury sustained. Meniscal pathology was addressed in 14 patients in both groups. Thirty-eight percent of all patients lacked >15° of knee flexion. Only one gross failure occurred, in the AG. No differences were noted in infection or re-operation. Lysholm Knee Scale and Tegner Activity Scale were not significantly different, although Tegner scores in both cohorts decreased from pre-injury scores. Conclusions: In summary, Artelon appears to be safe without increasing risk for hypersensitivity or infection when used for collateral ligament augmentation. Additionally, Artelon appeared to be non-inferior and statistically equivalent to primary repair in this setting and may have promise with use in certain types of knee dislocations.

Antibacterial agents that inhibit lipid A biosynthesis.

Lipid A constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and is essential for bacterial growth. Synthetic antibacterials were identified that inhibit the second enzyme (a unique deacetylase) of lipid A biosynthesis. The inhibitors are chiral hydroxamic acids bearing certain hydrophobic aromatic moieties. They may bind to a metal in the active site of the deacetylase. The most potent analog (with an inhibition constant of about 50 nM) displayed a minimal inhibitory concentration of about 1 microgram per milliliter against Escherichia coli, caused three logs of bacterial killing in 4 hours, and cured mice infected with a lethal intraperitoneal dose of E. coli.

Mechanisms and sequelae of E-cadherin silencing in hereditary diffuse gastric cancer.

Around 25-40% of cases of hereditary diffuse gastric cancer (HDGC) are caused by heterozygous E-cadherin (CDH1) germline mutations. The mechanisms for loss of the second allele still remain unclear. The aims of this study were to elucidate mechanisms for somatic inactivation of the wild-type CDH1 allele and to seek evidence for cadherin switching. Archival tumour material was analysed from 16 patients with CDH1 germline mutations and seven patients fulfilling HDGC criteria without CDH1 germline mutations. The 16 CDH1 exons were sequenced. E-cadherin promoter methylation was analysed by bisulphite sequencing and pyrosequencing and allele specificity was determined using polymorphic loci. Loss of heterozygosity was analysed using microsatellite markers. Cadherin expression levels were determined by real-time RT-PCR and immunohistochemistry. Six of 16 individuals with germline mutations had at least one second hit mechanism. Two exonic mutations (exon 9 truncating, exon 3 missense) and four intronic mutations which may affect splicing were identified. Tumours from 4/16 individuals had promoter hypermethylation that was restricted to the A allele haplotype in three cases. E-cadherin loss (mRNA and protein) generally correlated with identification of a second hit. In cases without germline E-cadherin mutations there was no evidence for somatic mutation or significant promoter methylation. P-cadherin (>25% cells) was expressed in 7/13 (54%) and 4/5 (80%) with and without germline CDH1 mutations, respectively, independent of complete E-cadherin loss. Overall, inactivation of the second CDH1 allele occurs by mutation and methylation events. Methylation is commonly allele-specific and is uncommon without germline mutations. P-cadherin over-expression commonly occurs in individuals with diffuse type gastric cancer.

A novel mutation in the E-cadherin gene in the first family with hereditary diffuse gastric cancer reported in Spain.

AIMS: Mutations of the E-cadherin gene (CDH1) result in dominantly inherited hereditary diffuse gastric cancer (HDGC). We report a study in the first family diagnosed with HDGC in Spain, examining the presence of mutations in the CDH1 gene. METHODS: The presence of mutations was studied by direct sequencing of all CDH1 exons. Immunohistochemical analysis with specific antibodies was used to detect the expression of E-cadherin in normal and tumour tissue. RESULTS: A novel 1610delC mutation in exon 11 has been found in a Spanish family diagnosed with HDGC. This mutation generates a premature stop codon at position 1667 giving rise to a truncated protein that lacks the transmembrane and beta-catenin-binding domains. The presence of a 1610delC germline mutation was confirmed in three family members diagnosed with diffuse gastric cancer, and also in six asymptomatic members. Of note, the diffuse gastric cancer coexisted with a gastric lymphoma in the proband. Furthermore, immunohistochemical analyses of tumour tissue showed the complete absence of E-cadherin in the proband, revealing a second genetic hit at the CDH1 locus. CONCLUSIONS: We have identified a HDGC family in Spain that carries a novel germline truncating mutation in the CDH1 gene.

The extension-adduction test in chronic tennis elbow: Soft tissue components and joint biomechanics.

Lateral elbow pain in 25 patients with chronic tennis elbow was reproduced by passive extension-adduction (EA) tests. Standard EA tests were performed with the addition of passive wrist flexion and extension and also with the forearm in the pronated position. These modifications were chosen in order to evaluate the effect on pain of a change in the lateral articular and extra-articular tissues. Performing the EA test in pronation rather than supination produced no significant change. However, both wrist flexion and extension.produced significant increases in pain. These results suggest increased tension in lateral structures relevant to tennis elbow and point to a useful addition to the standard EA test. The increase in elbow pain on wrist extension suggests that abnormalities of neural tension may contribute to pain. The results also indicate that a good comparable sign may be demonstrated with the forearm either pronated or supinated.

Rural health care issues in the Lower Mississippi Delta: an agenda for the year 2000.

The 214 counties bordering both sides of the Lower Mississippi Delta from Cape Girardeau, Missouri, to New Orleans, Louisiana, currently represent one of the four major poverty regions in the United States. Despite the location of five major medical centers, the Lower Mississippi Delta continues to lag behind the rest of the country in important health indices. This paper presents comparative data on infant mortality rates and health care availability between the Lower Mississippi Delta and the rest of the nation. It also contains comparative data on the rural Lower Mississippi Delta and the rest of the rural U.S. Further comparisons of European and African-American infant mortality rates reveal necessary directions and important realities for future health efforts. Commission research, gathered in public hearings from the Delta, reveals a pattern of interrelated development factors that also seriously affect health care in rural Delta counties.

Antibody levels and immunity to infectious bovine rhinotracheitis virus (IBR) infections in Wisconsin dairy cattle.

Individual cows and calves in 7 Holstein-Friesian herds with a history of respiratory disease in Green County, Wisconsin, USA, were swabbed intranasally and bled monthly, some for periods over 1 year. Virus isolation and serological procedures were completed on the samples in an attempt to recognize the problems and peculiarities that IBR virus continues to cause in dairy cows, in spite of, and possibly because of vaccines available for immunization against this agent. While IBR virus was isolated from animals on 6/7 farms over the period of study the history of vaccination with Nasalgen IP prevented the differentiation of vaccine (avirulent) virus from field (virulent) virus. IBR virus was isolated from apparently healthy animals as well as animals having signs of respiratory disease. IBR virus was assumed to be the cause of recurrent respiratory disease and ocular disease on at least two of the farms studied as established by virus isolation, serology, and the presence of characteristic signs of respiratory disease. While IBR virus was present on the other five farms its precise role in the diseases observed was not determined. IBR virus, whether vaccine virus or field virus, appeared to be persistent in 6/7 of the herds studied. Keratoconjunctivitis and corneal ulceration were the only signs of disease in animals in one herd where the etiologic agent was presumed to be field strain IBR virus. Antibody to IBR virus did not protect animals from respiratory or ocular disease in which IBR virus was thought to be a causative agent. Adult animals that had antibody to IBR virus at the time of vaccination with Nasalgen IP generally did not respond after vaccination with an increase in IBR virus antibody. Those adults that did not have antibody prior to vaccination, generally responded to vaccination with a rise in antibody. Vaccination of calves with Nasalgen IP generally did not cause an increase in antibody. IBR virus antibody was higher in vaccinated herds with recurrent respiratory disease.

Cloning, expression, and purification of UDP-3-O-acyl-GlcNAc deacetylase from Pseudomonas aeruginosa: a metalloamidase of the lipid A biosynthesis pathway.

The lpxC (envA) gene of Escherichia coli encodes UDP-3-O-acyl-GlcNAc deacetylase, the second and committed step of lipopolysaccharide biosynthesis. Although present in all gram-negative bacteria examined, the deacetylase from E. coli is the only example of this enzyme that has been expressed and purified. In order to examine other variants of this protein, we cloned the Pseudomonas aeruginosa deacetylase structural gene from a lambda library as a 5.1-kb EcoRI fragment. The LpxC reading frame encodes an inferred protein of 33,435 Da that is highly homologous to the E. coli protein and that possesses a nearly identical hydropathy profile. In order to verify function, we subcloned the P. aeruginosa lpxC gene into the T7-based expression vector pET11a. Upon induction at 30 degrees C, this construct yielded active protein to approximately 18% of the soluble fraction. We devised a novel, rapid, and reproducible assay for the deacetylase which facilitated purification of the enzyme in three steps. The purified recombinant protein was found to be highly sensitive to EDTA yet was reactivated by the addition of excess heavy metal, as was the case for crude extracts of P. aeruginosa. In contrast, deacetylase activity in crude extracts of E. coli was insensitive to EDTA, and the extracts of the envA1 mutant were sensitive in a time-dependent manner. The lpxC gene has no significant homology with amidase signature sequences. Therefore, we assign this protein to the metalloamidase family as a member with a novel structure.

Side effects of adjuvant chemotherapy: perceptions of node-negative breast cancer patients.

Twenty-one node-negative breast cancer patients were interviewed shortly after completing adjuvant chemotherapy and asked about side effects they had experienced, expectation of side effects, and strategies for coping with the side effects. Eighteen of the women were interviewed 6 months later to determine their feelings about the chemotherapy experience and ending treatment and what side effects persisted or developed after chemotherapy. Hair loss, fatigue, treatment-related problems, nausea and infections/low blood counts were the most frequently described problems during the first interviews. Patients used coping strategies suggested by physicians and nurses. Six months later, hair problems, fatigue, weight gain, menopausal problems, emotional problems and nail problems were most often reported. Most patients (16/18) did not expect to be experiencing chemotherapy-related problems 6 months after ending treatment. Fatigue interfered with daily lives and weight gain caused concern. A total of 35% of participants experienced fear or anxiety at the end of chemotherapy, but most (62%) recalled at least some positive feelings 6 months later. Given the same circumstances, all but two would make the same decision to undergo adjuvant chemotherapy. Support groups would be especially useful for patients completing chemotherapy who would lose continued frequent support from clinic personnel.

The envA permeability/cell division gene of Escherichia coli encodes the second enzyme of lipid A biosynthesis. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase.

The envA gene of Escherichia coli has been shown previously to be essential for cell viability (Beall, B. and Lutkenhaus, J. (1987) J. Bacteriol. 169, 5408-5415), yet it encodes a protein of unknown function. Extracts of strains harboring the mutant envA1 allele display 3.5-18-fold reductions in UDP-3-O-acyl-N-acetylglucosamine deacetylase specific activity. The deacetylase is the second enzymatic step of lipid A biosynthesis. The structural gene coding for the deacetylase has not been assigned. In order to determine if the envA gene encodes the deacetylase, envA was cloned into an isopropyl-1-thio-beta-D-galactopyranoside-inducible T7-based expression system. Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE. Direct deacetylase assays of cell lysates revealed a concomitant approximately 5,000-fold overproduction of activity. Assays of the purified, overproduced EnvA protein demonstrated a further approximately 5-fold increase in specific activity. N-terminal amino acid sequencing of the purified protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence. Contaminating species were present at less than 1% of the level of the EnvA protein. Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc deacetylase. Based on its function in lipid A biosynthesis, we propose the new designation lpxC for this gene.

Carbohydroxamido-oxazolidines: antibacterial agents that target lipid A biosynthesis.

A series of carbohydroxamido-oxazolidine inhibitors of UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase, the enzyme responsible for the second step in lipid A biosynthesis, was identified. The most potent analog L-161,240 showed an IC50 = 30 nM in the DEACET assay and displayed an MIC of 1-3 microg/mL against wild-type E. coli.

Isolation, biochemical characterization and ultrastructural analysis of the limbic system-associated membrane protein (LAMP), a protein expressed by neurons comprising functional neural circuits.

The limbic system-associated membrane protein (LAMP) is a cell surface glycoprotein expressed by cortical and subcortical regions of the mammalian CNS that comprise or receive direct projections from limbic system structures. The early and restricted expression of LAMP has led to its postulated role in neural development. Purification and biochemical characterization of LAMP was performed in order to ascertain its relationship to other, well-defined cell surface proteins in the nervous system. Subcellular fractionation, immunoaffinity chromatography, and Western blots of rodent and bovine hippocampus revealed that LAMP is an integral membrane protein with a molecular mass of 64-68 kDa and a pI of 5.2-5.5. Deglycosylation of LAMP indicates that it contains N-linked high mannose or hybrid sugars and a minor amount of sialic acid. The LAMP protein exhibits an identical molecular mass in developing hippocampus and in several different brain regions in the adult. No cross-reactivity was obtained using the monoclonal antibody that recognizes the HNK-1 carbohydrate epitope, a complex sulfated moiety expressed on members of a large family of glycoproteins. Immunocytochemical analysis at the ultrastructural level reveals that LAMP immunoreactivity is exhibited by neurons in a stereotyped pattern throughout limbic system areas. Glial cells are not immunoreactive. In the adult, LAMP-immunoreactive membrane patches are present exclusively postsynaptically on neuronal somata and dendrites. Myelinated and unmyelinated axons are not stained in any brain region examined. Analysis of LAMP expression in the developing CNS during synaptogenesis demonstrates that LAMP is located on growing axons and both pre- and postsynaptically at forming terminal complexes. Double-labeling studies of the hippocampal neurons grown in vitro reveal that the LAMP epitope is extracellular and is expressed on neurofilament- and microtubule-associated protein 2-positive neurites. Cells expressing glial fibrillary acidic protein are not LAMP-immunoreactive. These results demonstrate that in the adult brain, LAMP is expressed almost exclusively by the postsynaptic (target) elements in limbic circuits, but that during development, all components of the surface of the growing neuron contain LAMP. The stereotyped anatomical pattern of expression of LAMP in the developing and mature brain and its biochemical characteristics suggest that LAMP is a unique, system-associated membrane glycoprotein that is distinct from previously identified, developmentally important cell surface proteins.